Homogeneous, bioluminescent protease assays: Caspase-3 as a model
Homogeneous, bioluminescent protease assays: Caspase-3 as a model
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DOI：
10.1177/1087057104271865
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发表时间：
2005-03-01
影响因子：
--
通讯作者：
Wood, KV
中科院分区：
文献类型：
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作者：
O'Brien, MA;Daily, WJ;Wood, KV
使用caspase-3作为模型，作者开发了一种适用于高通量自动化应用的高灵敏度，均相蛋白酶测定的策略。该测定使用肽缀合的氨基荧光素作为蛋白酶底物和萤火虫荧光素酶，该荧光素酶已在分子上进化以增加稳定性。通过将促发光半胱天冬酶-3底物Z-DEVD-氨基荧光素与稳定的荧光素酶以均匀的形式结合，作者开发了一种比荧光半胱天冬酶-3测定法更快、更灵敏的测定法。该测定具有单步形式，其中底物的蛋白酶切割和氨基荧光素的荧光素酶氧化同时发生。由于这些过程是耦合的，它们迅速达到稳定状态，以保持稳定的发光数小时。当这种稳定状态发生时，达到最大灵敏度;因此，这种偶联酶系统导致非常快速的测定。新的均质形式固有地去除了游离氨基荧光素的痕量污染，导致极低的背景，并产生异常高的信噪比和优异的Z'因子。发光形式的另一个优点是它避免了可能与合成化学和天然产物文库相关的细胞自发荧光或荧光干扰的问题。这种生物发光，均匀的格式应广泛适用于其他蛋白酶测定。
Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease substrate and a firefly luciferase that has been molecularly evolved for increased stability. By combining the proluminescent caspase-3 substrate, Z-DEVD-aminoluciferin, with a stabilized luciferase in a homogeneous format, the authors developed an assay that is significantly faster and more sensitive than fluorescent caspase-3 assays. The assay has a single-step format, in which protease cleavage of the substrate and luciferase oxidation of the aminoluciferin occurs simultaneously. Because these processes are coupled, they rapidly achieve steady state to maintain stable luminescence for several hours. Maximum sensitivity is attained when this steady state occurs; consequently, this coupled-enzyme system results in a very rapid assay. ne homogeneous format inherently removes trace contamination by free aminoluciferin, resulting in extremely low background and yielding exceptionally high signal-to-noise ratios and excellent Z' factors. Another advantage of a luminescent format is that it avoids problems of cell autofluorescence or fluorescence interference that can be associated with synthetic chemical and natural product libraries. This bioluminescent, homogeneous format should be widely applicable to other protease assays.